Despite the fundamental roles of the mitochondrial respiratory chain (MRC) in both cellular energy production and a number of cardiovascular, neurodegenerative and inherited metabolic disorders, many factors required for MRC formation are currently unknown. In fact, almost 20% of the approximately 1000 known human mitochondrial proteins remain completely uncharacterized. Here we propose to address the gap in our understanding of MRC formation by systematically identifying and characterizing novel MRC biogenesis factors. We have developed an integrative genomic strategy based on clues from evolutionary history, high- throughput gene expression and protein interaction studies to discover novel MRC genes. Experimental work on two of our prioritized genes, C1orf31 and C6orf57, has shown their requirement for MRC complex IV and II biogenesis, respectively. Remarkably, a recent sequencing study identified mutations in C1orf31 in a mitochondrial disease patient. Due to the immediate relevance of C1orf31 to human health, we focused on characterizing the function of this protein in a yeast model where we demonstrated that copper supplementation rescued mitochondrial respiratory defects. In the current proposal we aim to: (1) Determine the role of C1orf31 in MRC complex IV assembly; (2) Investigate the pathological consequences of the loss of C1orf31 at the mitochondrial, cellular, and organismal level and determine the pathogenicity of patient mutations; and (3) Identify additional MRC biogenesis factors using our novel RNAi-based nutrient-sensitized assay that utilizes differential growth of respiratory deficient human cells in glucose or galactose to interrogate mitochondrial respiration. We will perform in vitro biochemical experiments on purified C1orf31 and in vivo yeast genetic experiments to define the precise function of C1orf31 in MRC complex IV assembly. We will exploit our C1orf31 knockdown models in human cell lines and zebrafish embryos to simultaneously unravel the pathological consequences of lack of C1orf31 in mitochondrial, cellular, and organismal physiology, as well as test the hypothesis that these defects could be cured by copper supplementation. Finally, we will experimentally test our computationally predicted MRC biogenesis gene candidates, including C6orf57, for their role in cellular respiration using our nutrient-sensitized assay and assign hits to specific steps in the MRC biogenesis pathway. Thus, the impact of our work is both fundamental (elucidating basic mechanisms of MRC formation) and medical (providing the basis for molecular diagnosis of orphan mitochondrial disorders and a possible therapeutic option for patients with C1orf31 mutations).